Fig. 1: Identification of ZNF768 as a phosphoprotein destabilized upon RAS activation.
A Overview of the RAS signaling pathway. B RPE cells were incubated with or without 10% serum for 2 h and western blots were performed. C Cells were transduced to overexpress RASG12V with a tamoxifen inducible system. Cells were exposed to tamoxifen (100 µM) for 3 days and western blots were performed. D, E U87 cells were treated with D PD098059 (50 µM) or E Torin1 (250 nM) and western blots were performed. F, G U87 cells were pretreated with F PD098059 (50 µM, 4 h) or G Torin1 (250 nM, 12 h) and then treated with cycloheximide (1 µg/ml). The result presented is the average of n = 3 (PD098059) and n = 4 (Torin1) independent experiments. H U87 cells were treated with PD098059 (50 µM), Torin1 (250 nM), or a combination of both drugs for 3 and 1 h, respectively and western blots were performed. I, J U87 cells were transduced to knockdown I RICTOR or J RAPTOR and western blots were performed. K Cells prepared as described in (I, J) were treated with cycloheximide as described in (F). The results were produced from at least six independent biological replicates per conditions (n = 6). L Hela cells were treated with Akt inhibitor VIII (50 µM) for 24 h and western blots were performed. M U87 cells were treated with Akt inhibitor VIII (50 µM) for 12 h and then treated with cycloheximide as described in (F). The results presented is the average of two experiments (n = 2). N Hela cells were transduced to overexpress a control protein or myr-Akt and western blots were performed. O Cells described in (N) were transduced to express V5-ZNF768 and treated or not with MG132 (20 µM) for 3 h and western blots were performed. P Phosphosite analyses showing the number of MS records for each serine residue found in ZNF768. Q Structure of ZNF768 protein and phosphorylation motifs found in the CTD domain. R U87 cells were transduced to overexpress V5-ACTA1 or V5-ZNF768. Immunoprecipitations using an antibody raised against PXS*P were performed and protein analyzed by western blot. S, T V5-ZNF768 and H-RASG12V were overexpressed in Hela cells. Immunoprecipitations and western blots were performed as described in (R). The results presented is the average of three experiments (n = 3). U U87 cells were transduced to overexpress ZNF768 phosphomutants and western blots were performed. In all panels, data represent the mean ± SEM. In panel F, G and K, significance was determined by two-way ANOVA. In panel T, significance was determined by two-tailed, unpaired t test. Details about reproducibility are provided in the Statistics and reproducibility included in the Methods section. Source data are provided as a Source data file.
